The fields of chromatin biology and epigenetics have experienced explosive growth in the last several years. Much of this growth has been enabled by technical advances in genome sequencing and proteomics, and buoyed by genetic links to human health and disease. The epigenome is an additional layer of chemical information that acts on top of the genome and informs gene expression. The complex array of posttranslational modifications (PTMs) to histone proteins, which act as molecular spools for wrapping DNA, is a major epigenetic mechanism for controlling transcriptional programs. These enzyme-catalyzed histone modifications (e.g. (de)acetylation and (de)methylation) result in a unique set of chemical 'marks' that regulate chromatin function, largely through unknown mechanisms. We and others have proposed that combinatorial posttranslational modifications (PTMs) give rise to a histone 'code' or 'language', which is interpreted by enzyme complexes to mediate transcriptional responses. New information suggests that 'reader' domains, which are protein:protein interaction modules, act within enzyme complexes to function as molecular interpreters of this combinatorial PTM code. However, direct molecular evidence is scarce. Thus, there is tremendous need to understand the molecular mechanisms of this essential process. Here, we will employ a number of innovative approaches to investigate the existence of a functional histone code and how this epigenetic language is read to control gene expression. The impact of this work toward human health is substantial: There is mounting genetic evidence that mutations in chromatin 'reader' domains are directly linked to human disease, and there are recent reports that 'reader' proteins can be targeted for drug development. The results of our mechanistic work will directly inform drug development. Three aims are proposed: 1.) To elucidate the binding mechanisms of tandem-domain chromatin readers, 2.) To define the PTM state of nucleosomes recognized by multivalent chromatin reader proteins, and 3.) To determine how chromatin enzymes utilize multivalent protein modules to read the PTM code written in nucleosomes.